The Saccharomyces cerevisiae Rad1-Rad10 complex is a conserved, structure-specific endonuclease important for repairing multiple types of DNA lesions. Upon recruitment to lesion sites, Rad1-Rad10 removes damaged sequences, enabling subsequent gap filling and ligation. Acting at mid-steps of repair, the association and dissociation of Rad1-Rad10 with DNA can influence repair efficiency. We show that genotoxin-enhanced Rad1 sumoylation occurs after the nuclease is recruited to lesion sites. A single lysine outside Rad1's nuclease and Rad10-binding domains is sumoylated in vivo and in vitro. Mutation of this site to arginine abolishes Rad1 sumoylation and impairs Rad1-mediated repair at high doses of DNA damage, but sustains the repair of a single double-stranded break. The timing of Rad1 sumoylation and the phenotype bias toward high lesion loads point to a post-incision role for sumoylation, possibly affecting Rad1 dissociation from DNA. Indeed, biochemical examination shows that sumoylation of Rad1 decreases the complex's affinity for DNA without affecting other protein properties. These findings suggest a model whereby sumoylation of Rad1 promotes its disengagement from DNA after nuclease cleavage, allowing it to efficiently attend to large numbers of DNA lesions.

Department of Biology Masaryk University Brno 62500 Czech Republic

Department of Biology Masaryk University Brno 62500 Czech Republic National Centre for Biomolecular Research Masaryk University Brno 62500 Czech Republic International Clinical Research Center St Anne's University Hospital in Brno Brno 60200 Czech Republic

Department of Molecular Medicine Institute of Biotechnology University of Texas Health Science Center at San Antonio San Antonio TX 78229 USA

Department of Molecular Medicine Institute of Biotechnology University of Texas Health Science Center at San Antonio San Antonio TX 78229 USA Division of Radiation Biology Department of Radiation Oncology University of Texas Health Science Center at San Antonio San Antonio TX 78229 USA

Molecular Biology Program Memorial Sloan Kettering Cancer Center New York NY 10065 USA

Molecular Biology Program Memorial Sloan Kettering Cancer Center New York NY 10065 USA Programs in Biochemistry Cell and Molecular Biology Weill Cornell Graduate School of Medical Sciences New York NY 10065 USA

National Centre for Biomolecular Research Masaryk University Brno 62500 Czech Republic

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MUS81 cleaves TOP1-derived lesions and other DNA-protein cross-links

Sumoylation regulates the stability and nuclease activity of Saccharomyces cerevisiae Dna2

Sumoylation influences DNA break repair partly by increasing the solubility of a conserved end resection protein

A versatile scaffold contributes to damage survival via sumoylation and nuclease interactions